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Chromosome Landing Using an AFLPTWased Strategy

Chromosome walking and, more recently, chromosome landing experiments have been successfully used in higher plants to identify genes correlated with specific genetic 1oc1 (1,2; for a review, see ref. 3). In Arabidopsis thaliana, these strategies are mainly used for mapping recessive mu ...

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Transposon Tagging with AC/Ds in Arabidopsis

Transposon tagging provides an effective method of isolating plant genes by identifying a mutation tagged by a DNA insertion, and then using the DNA tag to isolate the mutant gene (reviewed m ref. I). The first examples of this approach in plants involved transposon tagging in maize and Antirrhinum ...

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Cloning Genes from T-DNA Tagged Mutants

Insertion mutagenesis has a major advantage over other types of mutagenesis in that it not only causes a disruption of the gene, but the insertion element serves as a vehicle for recovery of the flanking DNA. Two types of insertion mutagens used extensively in plants are T-DN As and transposons. Each of t ...

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Whole-Mount In Situ Hybridization in Plants

Messenger RNA in situ hybridization (ISH) can be defined as a technique for locating specific transcripts. This powerful technique opened the way to study the temporal and spatial patterns of expression of animal and plant genes in a morphological context and on a cell-to-cell basis. Most in situ ...

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Bacterial and Coelenterate Luciferases as Reporter Genes in Plant Cells

The study of gene regulation has been greatly enhanced by the use of reporter gene systems such as beta galactosidase (P-gal), neomycin phosphotransferase (APH11), chloramphenicol acetyl transferase (CAT), beta glucuronidase (GUS), and dihydrofolate reductase (DHFR). In the past s ...

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Exploring the Potential of Plant RNase P as a Functional Genomics Tool

As we trek into the uncharted territories of the genomic era, there is an urgency for the development of approaches for assigning functions to the multitude of uncharacterized genes. Although currently available knock-out methodologies could be used for uncovering the function of newly ...

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P-Glucuronidase Enzyme Histochemistry on Semithin Sections of Plastic-Embedded Arabidopsis Explants

Reporter genes are commonly used m prokaryotes and eukaryotes to measure promoter activity of a gene of interest Transgenic organisms have to be generated by transformation with a chimertc gene consisting of the respective promoter and the coding sequence of the reporter gene. The activi ...

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Maintaining Collections of Mutants for Plant Functional Genomics

As the plant genomics era progresses and post-genomic functional research rapidly expands, varied genetic resources of unprecedented power and scope are being developed. Partially by the mandate of public funding, these resources are being shared via stock centers and private labor ...

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Genetic Screens for Floral Mutants in Arabidopsis thaliana: Enhancers and Suppressors

The flower is a hallmark feature that has contributed to the evolutionary success of land plants. Diverse mutagenic agents have been employed as a tool to genetically perturb flower development and identify genes involved in floral patterning and morphogenesis. Since the initial studi ...

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Detection of mRNA Expression Patterns by Nonradioactive In Situ Hybridization on Histological Sections of Floral Tissue

Analysis of gene activity with high spatial resolution is a prerequisite for deciphering regulatory networks which underlie developmental programs. Over many years, in situ hybridization has become the gold standard for the identification of in vivo expression patterns of endoge ...

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The GUS Reporter System in Flower Development Studies

The β-glucuronidase (GUS) reporter gene system is an important technique with versatile uses in the study of flower development. Transcriptional and translational GUS fusions are used to characterize gene and protein expression patterns, respectively, during reproductive de ...

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A Floral Induction System for the Study of Early Arabidopsis Flower Development

Assessing the molecular changes that occur over the course of flower development is hampered by difficulties in isolating sufficient amounts of floral tissue at specific developmental stages. This is especially problematic when investigating molecular events at very early sta ...

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Fluorescence Activated Cell Sorting of Shoot Apical Meristem Cell Types

Growing tips of plants harbor a set of stem cells in structures called shoot apical meristems (SAMs) which provide cells for development of aboveground biomass. Despite a periodic differentiation of stem cell progenitors into leaves, the stem cell pool remains constant over time. Genetic a ...

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Gene Expression Analysis by Quantitative Real-time PCR for Floral Tissues

Real-time, or quantitative, reverse transcription polymerase chain reaction (qRT-PCR), is a powerful method for rapid and reliable quantification of mRNA abundance. Although it has not featured prominently in flower development research in the past, the availability of novel tech ...

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Identification of Arabidopsis Knockout Lines for Genes of Interest

Determining gene function through reverse genetics has been an important experimental approach in the field of flower development. The method largely relies on the availability of knockout lines for the gene of interest. Insertional mutagenesis can be performed using either T-DNA or t ...

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Misexpression Approaches for the Manipulation of Flower Development

The generation of dominant gain-of-function mutants through activation tagging is a forward genetic approach that complements the screening of loss-of-function mutants and that has been successfully applied to studying the mechanisms of flower development. In addition, the fun ...

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Next-Generation Sequencing Applied to Flower Development: RNA-Seq

Genome-wide study of gene expression, or transcriptome profiling, is critical for our understanding of biological functions, including developmental processes. Recent breakthroughs in high-throughput sequencing technologies have revolutionized gene expression p ...

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Next-Generation Sequencing Applied to Flower Development: ChIP-Seq

Over the past 20 years, classic genetic approaches have shown that the developmental program underlying flower formation involves a large number of transcriptional regulators. However, the target genes of these transcription factors, as well as the gene regulatory networks they con ...

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Live-Imaging of the Arabidopsis Inflorescence Meristem

The aboveground tissues of higher plants are derived from a small population of stem cells located at the shoot apex within a structure called the shoot apical meristem (SAM). The SAM not only includes the stem cells but also incorporates a region from which lateral organs arise. The SAM is therefore of ...

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Gene Regulatory Network Models for Floral Organ Determination

Understanding how genotypes map unto phenotypes implies an integrative understanding of the processes regulating cell differentiation and morphogenesis, which comprise development. Such a task requires the use of theoretical and computational approaches to integrate and ...

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